Fig. 1.

Undifferentiated Caco2 cells contain abundant nLDs and LAPS. A: Caco2 cells were treated with oleate (400 μM) for 24 h, immunostained with CCTα and PML primary followed by AlexaFluor-647 and AlexaFluor-594 secondary antibodies, respectively, and imaged by confocal microscopy. LDs were visualized with BODIPY 493/503 (bar, 5 μm). B: quantification of the average cross-sectional area of total, PML-, and CCTα-positive nLDs. Results show the mean and SD from 6-8 fields of cells (10–15 cells/field) from three separate experiments. Significance was determined by one-way ANOVA and Tukey’s multiple comparison. C: SRRF images of Caco2 cells treated with oleate (400 μM) for 16 h were immunostained for CCTα and PML, BODIPY 493/503 to visualize LDs and imaged by spinning disc confocal microscopy (0.1 μm sections; bar, 5 μm). Enlarged region shown in a and b are boxed (bar, 1 μm). D and E: TEM images of Caco2 cells treated with oleate (500 μM) for 8 h (bar, 1 μm). Enlarged regions are boxed (bar, 200 nm). F: Caco2 cells transiently expressing V5-Lipin1α and treated with oleate (500 μM) for 16 h were immunostained with PML and V5 primary and AlexaFluor-647 and AlexaFluor-555 secondary antibodies, respectively, plus BODIPY 493/503 (bar, 5 μm). The line coordinates for the RGB plot are in the merged image. G: Caco2 cells transiently expressing a GFP-DAG sensor and treated with oleate (500 μM) for 16 h were immunostained for PML plus LipidTox Red (bar, 5 μm). The line coordinates for the RGB plot are in the merged image. H: Caco2 cells were treated with oleate (500 μM for 16 h) and immunostained with SUMO and PML primary followed by AlexaFluor-647 and AlexaFluor-594 secondary antibodies, respectively, plus BODIPY 493/503 (bar, 5 μm). Arrows indicate SUMO-deficient LAPS. ∗∗P < 0.01. CCTα, CTP:phosphocholine cytidylyltransferase; LAPS, lipid-associated promyelocytic leukemia structure; nLD, nuclear lipid droplet; PML, promyelocytic leukemia; SUMO, small ubiquitin-related modifier.